EGFR (T790M C797S), DEL 746-750, GST-Tag Recombinant
Catalog #
79434
$410*
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Description
Recombinant human EGFR (epidermal growth factor receptor, also known as HER1), encompassing amino acids 668-end and containing the tyrosine kinase domain. The protein contains deletion 746-750 and mutations T790M and C797S. This construct contains an N-terminal GST-tag. The recombinant protein was affinity purified and is kinase-active.
50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerol
MW
79 kDa + glycans
Amino Acids
668-end
Glycosylation
This protein runs at a higher MW by SDS-PAGE due to glycosylation.
Specific Activity
17 pmol/min/µg
Genbank #
NM_005228
UniProt #
P00533
Tag(s)
N-terminal GST-Tag
Background
EGFR is the receptor for members of the EGF family and is a transmembrane glycoprotein that has tyrosine kinase activity. Binding of epidermal growth factor to EGFR induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation, differentiation, motility, and cell survival (1). Activation of EGFR triggers mitogenic signaling in gastrointestinal mucosa, and its expression is upregulated in colon cancers and most neoplasms (2). Activation of EGFR triggers activation of the ERK-signaling pathway in normal gastric epithelial and colon cancer cell lines. Inactivation of EGFR with selective inhibitors significantly reduces ERK2 activation, c-fos mRNA expression and cell proliferation.
References
1. Wang, K., et al., J Biol. Chem 2004;279:53848-53856.
2. Kobayashi, S., et al., New Eng. J. Med. 2005;325:786-792.
Storage/Stability
At least 6 months at –80°C.
Assay Conditions
Kinase activity was measured using ADP-Glo™ Kinase Assay kit (Promega; Cat# V9101) which quantifies the amount of ADP produced by phosphorylation. The ADP-Glo™ Reagent is added to terminate the reaction and quench the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP and to measure the newly converted ATP using a luciferase reaction. Assay: Kinase activity was measured using substrate poly-Glu,Tyr 4:1 (stock 1 mg/ml). Increasing amounts of EGFR were incubated with a final concentration of 200 µg/ml substrate in 60 mM Tris-HCl, pH 7.4, 50 mM MgCl2, 4 mM MnCl2, 250 µg/ml BSA, 50 µM fresh DTT, and 25 µM ATP. The total incubation time was 40 min at room temperature. The reaction was terminated by addition of an equal volume of the ADP-Glo™ Reagent supplemented with 10 mM MgCl2, and the Kinase Detection Reagent was added. Phosphorylation was measured by detection of luminescence. The blank was determined from a “no kinase” sample.
Instructions for Use
Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.
Applications
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
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